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. 2020 Mar 10;295(17):5614–5625. doi: 10.1074/jbc.RA120.012588

Figure 4.

Figure 4.

Equilibrium binding of SC(1–246)-BODIPY and WT SC(1–246) to ProTQQQ. A, C, E, and G, SC(1–246)-BODIPY (29 nm (○) and 502 nm (●)) titrated with ProTQQQ, four separate batches. B, titration of 29 nm SC(1–246)-BODIPY (○); a mixture of 50 nm SC(1–246)-BODIPY and 50 nm WT SC(1–246) (▵); 502 nm SC(1–246)-BODIPY (●); and a mixture of 502 nm SC(1–246)-BODIPY and 500 nm WT SC(1–246) (▴) with ProTQQQ. D, F, and H, titrations of SC(1–246)-BODIPY (29 nm (○) and 502 nm (●)) and a mixture of 502 nm SC(1–246)-BODIPY and 500 nm WT SC(1–246) (▴) with ProTQQQ. Titrations shown in B, D, F, and H were performed with the corresponding, separate ProTQQQ preparations as in A, C, E, and G. The SC(1–246)-BODIPY titration data were analyzed by the quadratic binding equation to obtain the affinity, stoichiometry, and maximum fluorescence intensity (ΔFmax/Fo). Titration data of the probe and competitor were analyzed simultaneously by the cubic binding equation to obtain the affinity and stoichiometry of the competitor, WT SC(1–246) (Table 1).